Composition and manipulation of DNA molecules are important tasks for molecular biology in both research and industrial applications. While the problem of de novo DNA composition has been addressed systematically (5,10-12,16,17), no general method for editing DNA molecules is available, and specific editing tasks are presently addressed by specialized labor-intensive methods such as site-directed mutagenesis (1-3) and/or methods relying on the use of restriction enzymes (6, 7).
Biology labs engage daily in manual labor-intensive DNA processing—the creation of variations and combinations of existing DNA—using a plethora of methods such as site-directed mutagenesis (1,2,3), error-prone PCR (4), assembly PCR (5), cleavage and ligation (6,7), homologous recombination (8,9), and others (10,11,12,13,14,15). So far no uniform method for DNA processing has been proposed and, consequently, no engineering discipline has been able to eliminate the manual labor associated with DNA processing.
DNA composition, also called de novo DNA synthesis, is typically achieved by assembling synthetic oligonucleotides into ever longer pieces using one of several methods (17, 18). Although much progress has been made in achieving uniform, efficient and automated methods for performing de novo DNA synthesis, for example by avoiding cloning steps (17), while others use DNA microchips to reduce costs and errors (18) which occur very frequently in these de novo synthesis methods, still these methods suffer from many drawbacks. DNA editing, on the other hand, has no systematic solution to date, and the various editing tasks are performed by a plethora of labor-intensive methods (1-3). Site-directed mutagenesis generates targeted changes including single or multiple nucleotide insertions, deletions or substitutions, generally via the use of an oligonucleotide primer that introduces the desired modification. These fall into two major categories: those based on primer extension on a plasmid template (1-3), and PCR-based methods (13). Other methods use a restriction enzyme to cut the DNA molecule at specific preplanned sites of specific sequence and enable the ligation of a DNA fragment that contains the matching sites on its ends. In this method the short restriction sites must be specific and unique in the sequence to avoid undesired restrictions. Other methods generate random mutations using error-prone PCR, incremental truncation or random DNA shuffling. DNA shuffling performs in vitro homologous recombination of pools of selected homological DNA fragments by random fragmentation and PCR reassembly. However, this methodology is inefficient if multiple non-random sequence manipulations are required, as it requires iterative stages of mutagenesis, cloning, sequencing and selection. Therefore, current DNA processing methods do not provide for seamless DNA editing and manipulations that allow for the abstraction and creation of custom made, designer DNA molecules.